The present invention is directed to the gene TSC1 and to the protein it encodes. TSC1 has the characteristics of a tumor suppressor gene and is often mutated in individuals with a familial form of tuberous sclerosis. The invention also encompasses methods that relate to the gene and gene product.
Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by the development of unusual tumor-like growths (hamartomas) in a variety of organ systems (Gomez, M. R., Ann. N.Y. Acad. Sci. 615:1-7 (1991); Kwiatkowski, et al., Arch. Dermatol. 130:348-354 (1994)). The development of cortical tubers in the brain (regions of abnormal cortical architecture with distinctive large neuronal cells) causes some of the most problematic clinical manifestations of TSC: mental retardation, epilepsy and abnormal behavioral phenotypes including autism and attention deficit-hyperactive disorder (Hunt, et a., J. Autism. Dev. Disorder 23:323-339 (1993); Smalley, et al., J. Autism. Dev. Disorder 22:339-355 (1992)). Other organ systems commonly involved in TSC include the skin, heart, and kidneys (Kwiatkowski, et al., Arch. Dermatol. 130:348-354 (1994)). The lesions seen are often pathognomonic of TSC and include facial angiofibromas, subungual fibromas, forehead plaque, Shagreen patches, cardiac rhabdomyomas and renal angiomyolipomas and cysts. Renal cell carcinoma also occurs at higher frequency and at an earlier age of onset in TSC patients than in normal individuals (Bjornsson, et al., Am. J. Path. 149:1201-1208 (1996); Cook, et al., J. Med. Genet. 33:480-484 (1996)). About one-third of TSC cases are familial with the remainder being sporadic, i.e., occurring in the absence of a family history of the disease.
Linkage of TSC to 9q34 was first reported in 1987 and this locus was denoted TSC1 (Fryer, et al., Lancet i:659-661 (1987)). Subsequent studies provided strong evidence for locus heterogeneity and led to the identification of 16p13 (denoted the TSC2 locus) as a second genomic region showing linkage in some TSC families (Kandt, et al., Exp. Neurol. 104:223-228 (1989)). Among families large enough to permit linkage analysis, approximately half show linkage to 9q34 and half to 16p13 (Janssen, et al., Hum. Genet. 94:437-440 (1994)).
The TSC2 gene has been isolated and found to consist of 41 coding exons distributed over 45 kb of genomic DNA. It has a message length of 6 kb and encodes several alternatively spliced transcripts and predicted proteins of 1784-1807 amino acids (Maheshwar, et al., Hum. Mol. Genet. 5:131-137 (1996); Xu, et al., Genomics 27:475-480 (1995)). The occurrence of inactivating germline mutations in TSC2 in patients with tuberous sclerosis and loss of heterozygosity (LOH) at the TSC2 locus in up to 50% of TSC-associated hamartomas support a tumor suppressor function for TSC2 (Carbonara, et al., Cancer 15:18-25 (1996); Sepp, et al., J. Med. Genet. 33:962-964 (1996)).
Although the TSC2 gene has been isolated and characterized, identification of the TSC1 gene on 9q34 has proven difficult for a number of reasons. Conflicting positional information has been generated by the analysis of meiotic recombination events in TSC families; large genomic rearrangements (e.g., translocations involving this region) have not been discovered; and several parts of the region are unstable in multiple cloning vectors. In addition, the region contains a number of different genes, any one of which could potentially be TSC1. The unambiguous identification of the TSC1 gene would represent a significant advance in several different respects. First, assays designed to identify mutations in TSC1 could be used to help diagnose this condition in patients exhibiting clinical manifestations suggesting that they may suffer from tuberous sclerosis. Similarly, such assays could be used to help identify patients likely to develop the disorder or likely to pass it on to their offspring.
Therapeutically, the identification of the TSC1 gene and its product may provide a tool for the treatment of abnormal cellular growth. In the case of tuberous sclerosis patients, the introduction and expression of the normal TSC1 gene in place of mutated counterparts should help to prevent the development of hemartomas associated with the disease. In addition, the ability to inhibit uncontrolled cellular growth suggests that TSC1 and its product may have therapeutic applications for other conditions characterized by neoplastic growth.
The present invention is based upon the discovery of a gene (TSC1) which is found within the 9q34 region of chromosome 9. Mutations in TSC1 are associated with familial forms of tuberous sclerosis and the encoded protein, hamartin, has the characteristics of a tumor suppressor.
In its first aspect, the invention is directed to a protein, except as existing in nature, having an amino acid sequence consisting essentially of the sequence shown in FIG. 4 (SEQ ID NO:2). The term xe2x80x9cconsisting essentially of,xe2x80x9d is meant to encompass proteins having exactly the same amino acid sequence as shown in the figure, as well as proteins with differences that are not substantial as evidenced by their retaining the basic, qualitative functional properties of hamartin. The phrase xe2x80x9cexcept as existing in naturexe2x80x9d encompasses substantially purified forms of the protein as well as forms made by recombinant or synthetic means. A xe2x80x9csubstantially purifiedxe2x80x9d protein is one that has been separated from other accompanying biological components and will typically comprise at least 85% of a sample, with greater percentages being preferred. Many means are available for assessing the purity of a protein within a sample including analysis by polyacrylamide gel electrophoresis, chromatography and analytical centrifugation.
The invention also encompasses antibodies that bind specifically to hamartin (i.e., that have at least a 100-fold greater affinity for hamartin than any other undenatured protein), and antibodies made by a process involving the injection of a pharmaceutically acceptable preparation of hamartin into an animal capable of antibody production. In a preferred embodiment, monoclonal antibody to hamartin is produced by injecting the pharmaceutically acceptable preparation into a mouse and then fusing mouse spleen cells with myeloma cells.
The invention is also directed to a polynucleotide, except as existing in nature, which encodes a protein consisting essentially of the amino acid sequence of FIG. 4 (SEQ ID NO:2), expression vectors comprising such polynucleotides, and host cells transformed with such vectors. Also included is the human hamartin protein produced by host cells made in this manner. Preferably, the polynucleotide encoding the human hamartin has the sequence shown in FIG. 4 (i.e., that it comprises nucleotides 222-3713 of SEQ ID NO:1). It is also preferred that the vectors and host cells prepared for the expression of hamartin use this particular polynucleotide.
In another aspect, the present invention is directed to a method for identifying a patient that has, or is likely to develop, tuberous sclerosis by determining if the TSC1 gene of the patient is mutated, i.e., by determining whether it contains nucleotide additions, substitutions, or deletions relative to the wild-type TSC1 sequence. The patients examined by this method will typically be those that are exhibiting clinical characteristics suggesting that they may have tuberous sclerosis or individuals that have family members diagnosed as having the disease. In the first case, the method will be used in order to make or confirm a diagnosis and, in the latter case, it will be used to predict whether the patient or their offspring are likely to develop the disease. In general, the likelihood of a patient having or developing tuberous sclerosis increases in proportion to the number and seriousness of TSC1 mutations. Serious mutations include those that cause a frameshift resulting in the misreading of subsequent amino acids or that correspond to specific mutations that have been associated with tuberous sclerosis (see e.g., Table 4).
Any means may be used for determining the extent to which the TSC1 gene has been mutated, including direct nucleotide sequence analysis or hybridization under conditions selected to reveal mutations. However, the preferred method is to amplify one or more regions of the TSC1 gene using the polymerase chain reaction (PCR) and to then analyze the amplification products, e.g., by sequence analysis, heteroduplex analysis, or single strand conformation polymorphism analysis. In a preferred embodiment, the region amplified corresponds to one or more of exons 3-23, with the most preferred exons being 7, 9, 10, 15, 17, 18, 19 and 20.
An analysis of the characteristics of the hamartin protein indicates that it falls within the class of proteins that have been termed xe2x80x9ctumor suppressors.xe2x80x9d Mutations in these proteins which lead to a loss of function are oncogenic and it is recognized that the wild-type forms of the proteins suppress abnormal cellular proliferation. Examples of genes that have been found to encode tumor suppressors include the retinoblastoma susceptibility gene, the p53 gene, and the neurofibromatosis type 1 gene (Weinberg, R. A., Science 254:1138-1146 (1991)). Thus, the present invention is directed to a method for treating or preventing the abnormal proliferation of mammalian cells by introducing into the cells DNA encoding hamartin. The DNA must be incorporated into the cells in such a manner that it will undergo expression. In general, this means that the gene must be either operably linked to a promoter that is introduced at the same time as the gene itself or homologous recombination must be used to position the gene downstream from an endogenous promoter. This method may be carried out either on cells in vitro or in vivo. In either case, it is preferred that the cells be selected because they have a mutated form of the TSC-1 gene characteristic of tuberous sclerosis. The most preferred cells for use in conjunction with the method are those containing mutations in exons 3-23, and especially those with mutations in one or more of 7, 9, 10, 15, 17, 18, 19 and 20.